In the field of clinical laboratory test, classifying and counting leukocytes with abnormal DNA amount or immature leukocytes provide very useful information in diagnosis of diseases. For example, leukocytes in normal peripheral blood generally consist of these five-types, i.e., lymphocytes, monocytes, basophiles, eosinophils and neutrophils, and the DNA amount of leukocytes is constant. However, leukocytes with abnormal DNA amount appear, for example, in blood disorders such as viral diseases or lymphocytic leukemia, and immature leukocytes appear in such blood disorders as myelocytic leukemia.
Usually, in order to measure leukocytes with abnormal DNA amount, it is necessary to mix and stain mononuclear leukocytes (lymphocytes and monocytes), which are separated from leukocytes by specific gravity centrifugation, or peripheral blood with a nucleic acid-staining dye and a hemolytic agent, and subsequently to carry out the measurement. Separation of the mononuclear leukocytes by specific gravity centrifugation is complicated in operation and requires 1 hour or more for completion of the separation. In addition, 30 minutes or more are required for mixing and staining mononuclear leukocytes or peripheral blood with the nucleic acid-staining dye and the hemolytic agent.
In general, in order to classify and count immature leukocytes, a blood smear is prepared, properly stained and observed under a microscope for classification and count. On the other hand, in recent year, a variety of full-automatic hematocytometers have been provided utilizing the principle of a flow cyctometer. Although normal leukocytes can be highly precisely classified and counted with these cytometers, immature leukocytes can not precisely be detected and classified because the cytometers are greatly influenced by platelet clumps and coincidence cells.
On the other hand, it has been reported that immature leukocytes and normal leukocytes can be simultaneously classified and counted by maintaining the immature leukocytes in a viable state and treating other leukocytes with a hemolytic agent which damages other leukocytes; staining the damaged cells with a fluorescent dye which can stain the damaged cells; and measuring scattered light and fluorescence of the resulting blood corpuscles (Japanese Unexamined Patent Application Hei 10(1998)-206423). In this method, however, it was not possible to differentiate leukocytes with abnormal DNA amount from platelet clumps and coincidence cells, thus it was difficult to precisely classify and count leukocytes with abnormal DNA amount.
Thus, there is a need for a rapid, simple and highly precise method for measuring leukocytes with abnormal DNA amount and simultaneously, immature leukocytes.